Atherosclerotic vascular disease is the leading cause of death among people with diabetes mellitus. One important mechanism may involve oxidative stress because elevated blood glucose is the hallmark of diabetes and glucose promotes glycoxidation reactions in vitro. Moreover, glycoxidation products accumulate in tissues of diabetic animals and humans. The overall goal of this research proposal is to identify the biochemical pathways through which glucose accelerates glycoxidative stress. We plan to investigate four specific questions. First, we will determine whether diabetic hyperglycemia produces a distinct pattern of oxidation products in human atherosclerotic lesions. Our GUMS analyses of vascular tissue isolated from diabetic primates strongly suggests that hyperglycemia creates localized oxidative stress in the artery wall. To determine whether glucose-enhanced oxidative pathways also contribute to human atherosclerosis, we will use mass spectrometry to quantify glycoxidation products in vascular tissue. Second, we will identify the patterns of protein oxidation products that arise when peptides are exposed to reactive intermediates implicated in diabetic vascular disease. This arm of the project will complement our in vivo work by identifying markers of biochemical pathways that are triggered by hyperglycemia and that also oxidize proteins and LDL in vitro. Third, we will use mouse models to determine the role of glycoxidation in protein oxidation, AGE formation, and the development of diabetic vascular disease. Finally, we will use cultured human macrophages to identify glycoxidized proteins and proteins whose expression is markedly affected by hyperglycemia. These experiments should identify intracellular proteins that are vulnerable to glycoxidation. They will also directly test the hypothesis that glucose-stimulated superoxide production by mitochondria regulates macrophage gene expression.